The European purple sea urchin Paracentrotus lividus is a keystone species of benthic coastal communities and an important marine resource in southern Europe (Bertocci et al., 2018). The growing interest in increasing aquaculture production has been driving the research for diets able to supply species nutritional requirements and produce high quality roe. A well-balanced diet can also contribute to maintaining an efficient immune system. Like other invertebrates, the sea urchins cannot biosynthesize carotenoids de novo (Tan et al., 2020), but these are directly involved in the immune and antioxidant defense system. The carotenoids are functional nutrients that stimulate the activity of antioxidant enzymes, increasing the efficiency of detoxification after immune activity (Babin et al., 2015). Simultaneously, by taking over the antioxidant enzyme activity and eliminating free radicals, carotenoids reduce the need for these enzymes and overall burden (Tan et al., 2020). A high dietary carotenoid content enhances immunity by supporting endogenous enzymes (catalase and superoxide dismutase) and detoxifying free radicals produced during immune activity. Despite the important physiologic role, In sea urchins, coelomocytes are the main cellular components responsible for the immune response (Banks, 2014) seconded by the proteases and lysozymes, representing a barrier against bacterial proliferation due to their peptidase activity (Fernández-Boo et al., 2018). The main objective of the present study was to analyze the effect of high and low carotenoid levels on the immune and oxidative response of P. lividus sea urchins.
Material and Methods
Adult sea urchins (N = 153) with mean diameter of 38.57 ± 2.99 mm were collected on a rocky beach (Porto Batel, Peniche) and maintained in three recirculatory aquatic systems (RAS) equipped with three 40 L glass tanks each (N = 17 individuals/tank). For 8 weeks, the animals were fed three times a week with three jellified diets: a diet rich in carotenoids (HC); a diet low in carotenoid (LC); seaweed-based control diet (Control). The high content of carotenoids in the diets was accomplished by adding pumpkin and dried Nannochlropsis sp to a vegetable-based diet. At the end of the nutritional trial, the sea urchins were exposed to a time-course pathology challenge, where they were inoculated with Vibrio parahemolyticus to stimulate the individual’s immune system. Sea urchin’s weight, condition, immune and oxidative status were measured at the beginning and at the end of the nutritional trial. In detail, the sea urchins were measured and weighed, their gonads were removed and weighed to determine gonadosomatic index and to quantify the activity of oxidative stress related enzymes (Catalase – CAT; Superoxide dismutase - SOD; Lipid peroxidation - LPO). For the analysis of cellular and humoral immune parameters, coelomic fluid (CF) was collected individually to identify and count coelomocytes under 400x magnification microscope. For the analysis of humoral parameters were used the turbidimetric assay to quantify the lysozyme concentration, the azocasein hydrolysis assay for protease activity (Fernández-Boo et al., 2018) and to quantify the nitric oxide content, the Griess reaction procedure (Tafalla et al., 2003).
In the end of nutritional trial, there was an increase of coelomocytes in the sea urchins fed with HC and LC diets ( respectively) when compared to the initial value (), while in the control diet there was a decrease in the total number of coelomocytes (). From these coelomocytes, and similarly in the three dietary groups, the most abundant were the phagocytes (HC: 58%; LC: 65%; Control: 69%), followed by the colorless granulocytes (HC: 18%; LC: 17%; Control: 16%). For the humoral parameters, the lysozyme (HC: 0.86 µg/mL; LC: 0.82 µg/mL; Control: 0.42 µg/mL) and protease (HC: 13%; LC: 14%; Control:13%) did not differ between the three diets studied. On the other hand, remarkable differences in the parameters of oxidative stress were observed between dietary treatments. The production of CAT was higher in individuals fed with HC diet (42 U mg/protein) and LC diet (54 U mg/protein) in relation to those fed with control diet. The opposite occurred with the production of SOD. Here, the animals fed with control diet produced a high amount of this enzyme (131 U/mg), compared with animals fed LC diet (50 U/mg). The occurrence of lipid peroxidation was more visible on sea urchins belonging to the treatment with the LC diet (0.043 nmol/g wt).
The cellular and humoral immune parameters analyzed in the sea urchins CF were not affected by the dietary carotenoids’ levels selected. Regarding the antioxidant defense system, there was an effect of the carotenoids included in the diets provided to the sea urchins. The high content of carotenoids in an organism, as part of the integrated antioxidant system, enhances immunity by assisting endogenous enzymes (for example, catalase and superoxide dismutase) (Tan et al., 2020). Peroxidation is a common cellular damage caused by oxidative stress, because of the reaction of ROS with lipids. However, the effect of this cellular damage can be minimized by the activation of cytoprotective enzymes, such as superoxide dismutase (SOD) and catalase (CAT) (Amorim et al., 2020). In the present study, the urchins fed with LC diet presented low SOD, higher CAT values and then higher lipid peroxidation values than the HC and Control diet. This could mean that the production of catalase wasn’t enough to prevent the occurrence of lipid peroxidation in this treatment.
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